The bacterium Pseudomonas aeruginosa is an opportunistic pathogenic organism which often occurs with hospital infections, mainly in patients having an impaired immune defense, such as patients suffering from burns, persons suffering from cystic fibrosis or having organic malfunctions, and in tumor patients. Due to the occurrence of resistances, antibiotics are active against Pseudomonas infections only to a limited extent and, therefore, it has been endeavored to find immunologic methods for fighting infections caused by Pseudomonas aeruginosa.
Infections may be triggered by a variety of strains that may be characterized by O-antigens (lipopolysaccharide) and H-antigens (flagella), H- and O-antigen types being freely combinable.
It is known that the motility of Pseudomonas aeruginosa attributes to the virulence of the pathogen (Montie et al., Infect. Immunity 38, 1296-1298, 1982). Motile Pseudomonas aeruginosa strains possess a single flagellum. Burned mouse model assays have revealed that a larger percentage of mice survive if non-motile Pseudomonas aeruginosa strains are inoculated in experimentally produced burns than do if motile strains are used (Montie et al., see above). Further assays on the pathogenesis of Pseudomonas aeruginosa demonstrated that animals that had been immunized with flaggelar antigen preparations were protected when setting a burn and infecting with motile bacteria strains (Holder et al., Infect. Immunity 35, 276-280, 1982).
Pseudomonas aeruginosa flagella were investigated by serological methods, and it was demonstrated that there may be differentiated between two main types of antigens denoted as type a and type b according to R. Ansorg (Zbl. Bakt. Hyg. I. Abt. Orig. A, 242, 228-238, 1978).
In flagellar (H) antigen type a, the partial antigens a.sub.0, a.sub.1, a.sub.2, a.sub.3 and a.sub.4 are differentiable by using the indirect immunofluorescence technique; they are expressed by the Pseudomonas aeruginosa strains in different combinations, the partial antigen a.sub.0 being found on all flagella of type a. Flagellar (H) antigen type b behaves serologically uniformly, i.e., no subgroups can be identified.
According to Ansorg, a total of 17 H-types may be differentiated with Pseudomonas aeruginosa, i.e., one H-type b and 16 H-types of flagellar antigen a, consisting of the partial antigen a.sub.0 present in all a-types and optionally of the partial antigens a.sub.1 and/or a.sub.2 and/or a.sub.3 and/or a.sub.4. Comprehensive studies of flagella of the Pseudomonas aeruginosa strains 5142 and 13030, which had been characterized by Ansorg as exclusively belonging to subgroup a.sub.0, proved the same to be biochemically and immunologically identical with flagella type b (Montie et al., Infect. Immunity 49(3), 770-774 (1985)).
The obtention of polydisperse native flagellar (H) antigens (FAgs) is described in PCT publication WO 86/03974. Bacterial cultures are grown in minimal medium and subsequently are treated with a detergent, whereupon FAgs are separated from the culture. Prior to the treatment with a detergent, the bacterial culture is disintegrated and subjected to a shearing process. By the addition of detergent, the separability of the antigen from the bacteria mass is promoted; the antigens then being in suspension can be separated from the cells by differential centrifugation.
Subsequently, the antigens separated from the bacterial mass as described above can be freed from adhering impurities, such as lipopolysaccharides, nucleic acids, salts, polysaccharides and the like by chromatographic purification. The detergent still present can be eliminated by an additional column-chromatographic purification step.
The strains used for preparing Pseudomonas aeruginosa bacterial cultures and the antigens produced are listed in the following Table.
______________________________________ Strain H-type ______________________________________ M-2 b 5142 a.sub.0 (b) 5940 a.sub.0 a.sub.2 WR-5 a.sub.0 a.sub.2 5939 a.sub.0 a.sub.3 1210 a.sub.0 a.sub.1 a.sub.2 170018 a.sub.0 a.sub.3 a.sub.4 ______________________________________
From EP-A-0 252 064 it may be taken that all individual flagellar antigens of the types described in PCT publication WO 86/03974, as components of a vaccine, exhibit protective action and that the serum obtained from mice that had been actively immunized with the flagellar antigens indicated in PCT publication WO 86/03974 is suited for the passive immunization of mice.
However, the protective effect of the individual antigens and of the antibodies derived therefrom was detectable only in the animal model and only in the homologous system, i.e., an activity was observed only against pathogens of that flagellar serotype which had been utilized for immunization.
On the physicians' part, there is the desire to have available a Pseudomonas aeruginosa vaccine whose efficacy is directed against any combination of the partial flagellar (H) antigens a.sub.0, a.sub.1, a.sub.2, a.sub.3, a.sub.4 and b, since an analysis of 300 clinical Pseudomonas aeruginosa isolates by the H-antigen serotyping scheme proposed by Ansorg (Zbl. Bakt. Hyg. I. Abt. Orig. A242, 228-238, 1978) revealed that partial antigens of types a and/or b were actually detected in 98% of all motile Pseudomonas pathogens.
According to the findings disclosed in EP-A-0 252 064, a vaccine of this kind must necessarily contain all these partial antigens, i.e., must be a polyvalent vaccine.